Vectors containing HIV packaging sequences, packaging defective HIV vectors, and uses thereof

ABSTRACT

Packaging defective and packaging proficient HIV vectors are disclosed. These vectors can be used to establish HIV packaging defective cell lines, and to package desired genes. These cell lines can be used in developing a vaccine, HIV antibodies and as part of a system for gene transfer. The packaging proficient vector can be used to target HIV target cells.

This is a divisional of application Ser. No. 08/152,902 filed Nov. 15,1993, now U.S. Pat. No. 5,665,577, which is a continuation of Ser. No.07/827,588 filed Jan. 29, 1992, now abandoned, which was a continuationof Ser. No. 07/540,746 filed Jun. 20, 1990, now abandoned.

The present invention is directed to vectors including vectorscomprising a packaging defective HIV provirus, a vector comprising a HIVpackaging sequence and a gene to be transferred, the use of thepackaging defective vectors to create HIV packaging defective celllines, and the uses of the vectors and cell lines. Most preferably, theHIV provirus is an HIV-1 provirus.

The human immunodeficiency virus (HIV-I, also referred to as HTLV-III,LAV or HTLV-III/LAV) is the etiological agent of the acquired immunedeficiency syndrome (AIDS) and related disorders [Barre-Sinoussi, etal., Science 220:868-871 (1983); Gallo et al, Science 224:500-503(1984); Levy et al., Science 225:840-842 (1984); Popovic et al., Science224:497-500 (1984); Sarngadharan et al., Science 224:506-508 (1984);Siegal et al., N. Engl. J. Med. 305:1439-1444 (1981)]. The disease ischaracterized by a long asymptomatic period followed by progressivedegeneration of the immune system and the central nervous system.Studies of the virus indicate that replication is highly regulated, andboth latent and lytic infection of the CD4 positive helper subset ofT-lymphocytes occur in tissue culture [Zagury et al., Science231:850-853 (1986)]. The expression of the virus in infected patientsalso appears to be regulated to enable evasion of the immune response.Molecular studies of the regulation and genomic organization of HIV-Ishow that it encodes a number of genes [Ratner et al., Nature313:277-284 (1985); Sanchez-Pescador et al., Science 227:484-492 (1985);Muesing et al., Nature 313:450-457 (1985); Wain-Hobson et al., Cell40:9-17 (1985)]. The other primate immunodeficiency viruses, HIV-2 andsimian immunodeficiency virus (SIV) also share many of the samestructural and regulatory genes such as gag, pol, env, tat, rev and nef[Guyader, M., et al., Nature 326:662-669 (1987); Chakrabarti, L., etal., Nature 328:543-547 (1987), which are incorporated herein byreference].

Retroviruses are typically classified as belonging to one of threesubfamilies, namely oncoviruses, spumaviruses and lentiviruses.Infection by oncoviruses is typically associated with malignantdisorders. These viruses typically contain a single-stranded,plus-strand RNA genome of approximately 8,000 to 10,000 nucleotidesencompassing the gag, pol and env, genes, as well as long terminalrepeat (LTR) sequences. Oncoviruses typically contain an oncogene. It isgenerally believed that spumaviruses are not pathogenic in vivo,although they induce foamy cytopathic changes in tissue culture.Infection by lentiviruses is generally slow and causes chronicdebilitating diseases after a long latency period. These viruses, inaddition to the gag, pol, and env genes possess a number of additionalgenes with regulatory functions.

The human immunodeficiency viruses (HIV) has been classified as alentivirus, because it too causes slow infection and has structuralproperties in common with such viruses. [See Haase, A. T., Nature 322:130-136 (1986)].

All known retroviruses share features of the replicative cycle,including packaging of viral RNA into virions, entry into target cells,reverse transcription of viral RNA to form the DNA provirus, and stableintegration of the provirus into the target cell genome [Coffin, J., J.Gen. Virol. 42:1-26 (1979)]. Replication-competent proviruses, at aminimum, contain regulatory long terminal repeats (LTRs) and the gag,pro, pol, and env genes which encode core proteins, a protease, reversetranscriptase/RNAse H/integrase and envelope glycoproteins, respectively[J. Gen. Virol. 42, supra]. The LTRs contain cis-acting sequencesimportant for integration, transcription and polyadenylation.

HIV shares the pg, pro, pol and env genes, respectively with otherretroviruses [Haseltine, W. A., Journal of Acquired Immune DeficiencySyndrome, 1:217-240 (1988)]. HIV also possesses additional genesmodulating viral replication. The HIV-1 genome encodes vif, vpr, tat,rev, vpu and nef proteins [Haseltine, W. A., Journal of Acquired ImmuneDeficiency Syndrome, supra]. Additionally, the long terminal repeats(LTRs) of HIV contain cis-acting sequences that are important forintegration, transcription and polyadenylation. Additional cis-actingsignals allow regulation of HIV sequences by some of the novel HIV geneproducts, (Haseltine, W. A., Journal of Acquired Immune DeficiencySyndrome, supra). Sodroski et al., Science 231:1549-1553 (1986); Arya etal., Science 229:69-73 (1985); Sodroski et al., Science 227:171-173(1985); Sodroski et al., Nature 321:412-417 (1986); Feinberg et al.,Cell 46:807-817 (1986) Wong-Staal et al, AIDS Res. and HumanRetroviruses 3: 33-39 (1987); which are all incorporated herein byreference]. The region between the 5' major splice donor and the gaggene initiation codon is highly conserved in different HIV-1 strainssequenced to date [Myers, G., et al, Theoretical Biology and Biophysics,(1988)].

Most of these genes encode products that are necessary for the virallife cycle. For example, the tat gene encodes a 14kD protein that iscritical for HIV replication and gene expression [Rosen, C. A., et al.,Nature 319:555-559 (1986); Sodroski, J. et al., Science 227:171-173(1985); Arya et al, Science 229: supra, Sodroski, et al., Science 229,supra and Dayton, A., et al., Cell 44:941-497 (1986) which are allincorporated herein by reference]. Another gene necessary forreplication is the rev gene. [Sodroski, et al., Nature 321:412-417(1986), which is both incorporated herein by reference].

In some oncoviruses, cis-acting sequences located between the 5' LTR andthe gag gene initiation codon have been located which are necessary forthe efficient packaging of the viral RNA into virions [Bender, M. A., etal, J. Virol 61:1639-1646 (1987), Katz, R. A., et al, J. Virol59:163-167 (1986), Mann, R., et al, Cell 33:153-159 (1983), Pugatsch,T., et al, Virology 128:505-511 (1983), Watanabe, S., et al, Proc. Natl.Acad. Sci. 79:5986-5990 (1983) Eglitis, M. A., et al, Bio Techniques6:608-614 (1988) which are incorporated herein by reference]. Inaddition to these sequences, sequences overlapping the gag gene werefound to contribute to the efficiency of viral RNA encapsidation byMoloney murine leukemia virus [Adam, M. A., et al, J. Virol.62:3802-3806 (1988); Bender, M., et al., J. Virol. 61:1639-1646 (1987)].Certain retroviruses have been used to introduce genetic informationstably into the genome of target cells in eukaryotic cells in vivo andin vitro [Cornetta, K., Progress in Nucleic Acids Research and MolecularBiology 36:311-322 (1989); Gilboa, E., Biotechniques 4:504-512 (1986);Joyner, A., Nature 305:556-558 (1983); Mann, R., et al., Cell 33:153-159(1983)]. Vectors containing the desired gene and packaging sequenceswere incorporated by packaging signal-deleted viruses generating virionscapable of entry into certain cells. The signals needed for packaging oflentiviruses RNA, (such as HIV RNA) into virion particles have not beenidentified.

Although a great deal of research has been expended on understandingHIV-1, the life cycle of this retrovirus is not completely understood.

In addition, a great deal of research has been directed to developing avaccine to the virus, but there have been no reports of success to date.This is, in part, due to the lack of conservation in the antigenicallyactive parts of the virus and in part because the functionally importantregions of viral proteins and/or inactivated viral particles are poorlyimmunogenic.

Many methods proposed for treating HIV infected individuals wouldadversely affect uninfected cells as well as HIV infected cells.

Accordingly, it would be extremely useful to have a provirus thatproduced HIV proteins but which was not lethal because the viral RNAcould not be packaged into virions. Using this packaging-defectiveprovirus vector, it would be possible to create packaging defective celllines that could be used to investigate the packaging mechanism of thevirus and to develop strategies to interfere with this packagingmechanism. Significantly, the virions produced by such packagingnegative proviruses could be used for vaccines and as a system forefficiently introducing a desired gene into a mammalian cell.

It would also be useful to have a vector that could be selectivelytargeted to HIV target cells and could thus introduce a desired productinto such cells.

SUMMARY OF THE INVENTION

We have now discovered a vector comprising a sufficient number ofnucleotides corresponding to an HIV genome to express functional HIVgene products (HIV nucleotides), but which does not contain a sufficientnumber of nucleotides corresponding to nucleotides of the HIV genomebetween the 5' major splice donor and the gag gene initiation codon toefficiently package the viral RNA into virions (HIV packaging sequence).Preferably, this HIV packaging sequence corresponds to the regionbetween the 5' major splice donor and the gag gene initiation codon(nucleotides 301-319). More preferably, this sequence corresponds to asegment just downstream of the 5' major splice donor, and about 14 basesupstream of the gag initiation codon. In one embodiment it is a 19 basesegment having the sequence AAAAATTTTGACTAGCGGA.

This vector can be used to transform a preselected cell line to resultin an HIV packaging defective cell line. Preferably, one would transforma cell line using at least two vectors, which collectively contain theHIV nucleotides necessary to express HIV gag, pol, and env products, butwherein each vector by itself does not contain the HIV nucleotidesnecessary to express all three products. In addition, each vector doesnot have a sufficient number of nucleotides corresponding to nucleotidesof the HIV genome between the 5' major splice donor and the gag gene toefficiently package HIV RNA. More preferably, each vector would notcontain a sequence corresponding to an LTR sequence downstream of thenucleotides corresponding to the HIV genes. Preferably, each vectorwould contain a different marker gene. The transformed cell line wouldexpress HIV virions but would not be able to package HIV RNA into thesevirions. Thus, these virions could be used to generate antibodies, as avaccine or as a method of transferring a desired gene product to adifferent cell line capable of infection by HIV.

A second vector contains a preselected gene, a sufficient number ofnucleotides corresponding to an HIV packaging sequence to package HIVRNA (HIV packaging sequence), and is flanked on each side with asequence corresponding to a sufficient number of HIV LTR nucleotides tobe packaged by the HIV packaging sequence (HIV LTR sequences), whereinthe HIV packaging sequence and HIV LTR sequences correspond to the sameHIV genome. This vector can be used with the packaging defective vectorsto transfer the desired preselected gene. Alternatively, the vector canbe administered to an HIV infected cell and be packaged by the HIVvirions being produced. The HIV infected cell can be in an individual.Combinations where the HIV packaging defective vector and HIV virus as ahelper virus are used together are also described. The packagingsequences are located in a region from the 5' major splice donor to asite within the 5' most part of the gag gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the HIV-1 genome from the 5' LTR to the gaginitiation codon showing the 5' major splice donor (SD) and the site ofthe deletion in a vector representing one embodiment of this invention,pHXBΔP1.

FIGS. 2a-e represents schematics of vectors representing variousembodiments according to this invention. FIG. 2a is a packagingdefective vector, HXBvP1. FIG. 2b is a packaging defective vector,HXBΔP1Δenv. FIG. 2c is a packaging defective vector, pSVIIIenv 3-2. FIG.2d is a packaging proficient vector, HVB(SL3-Neo). FIG. 2e is apackaging proficient vector, HVB(SL3-Neo).

FIG. 3 is a schematic of one preferred embodiment showing two packagingdeficient vectors, HXBΔP1Δenv and pSVIIIenv 3-2.

FIG. 4 is a chart showing p24 Levels in Culture of Infected Jurkat Cellsby vectors representing various embodiments of this invention.

FIG. 5a is an autoradiogram of the immunoprecipitation of ³⁵ S-labelledviral protein from COS-1 cells transfected with pHXBΔP1 DNA with AIDSpatient serum.

FIG. 5b is an electron micrograph of COS-1 cells transfected withpHXBΔP1 showing virion particles of normal HIV-1 morphology.

FIG. 6 is an autoradiogram of immunoprecipitation of labelled viralproteins from Jurkat T cell lysates or supernatants exposed tosupernatants from COS-1 cells that were transfected or mock transfected.

FIG. 7 is an RNA dot blot test.

FIG. 8 is an autoradiogram showing the Sourthern blot of total DNA ofG418-resistant Jurkat cells.

DETAILED DESCRIPTION OF THE INVENTION

We have now discovered that it is possible to make HIV packagingdefective vectors and cell lines. We have found that the region betweenthe 5' major splice donor and the gag gene initiation codon in HIVviruses contains sequences necessary for packaging of HIV RNA intovirions. One can prepare a vector comprising a packaging defective HIVprovirus wherein the vector contains a nucleotide sequence whichcorresponds to a sufficient number of nucleotides from an HIV genome toexpress desired HIV products, but does not correspond to a sufficientnumber of nucleotides corresponding to the region between the 5' majorsplice donor and the gag gene initiation codon to efficiently packageHIV RNA (the HIV packaging sequence).

These sequences preferably correspond to the genome of HIV-1, HIV-2 andsimian immunodeficiency virus (SIV). [See Ratner, et al, Nature 313,supra, Sanchez-Pescador et al, Science 227, supra, Muesing, et al,Nature 313, supra, Wain-Hobson et al, Cell 40, supra, Guyader, M. et al,Nature 326, supra (1987); Chakrabarti et al, Nature 328, supra (1987)and Hirsch, V., et al, Cell 49:307-319 (1987) which are all incorporatedherein by reference].

The term corresponding means that conservative additions, deletions andsubstitutions are permitted.

Preferably, the vector does not contain the HIV packaging sequencecorresponding to the segment immediately downstream of the 5' majorsplice donor and just upstream of the gag gene initiation codon.Typically, the vector could contain nucleotides ranging from about 14bases to 2 bases upstream of the gag initiation codon (for exampleeither the 14 upstream bases or 5 upstream bases) and still be packagingdeficient. In one embodiment the vector does not contain a nucleotidesequence beginning about 9 bases downstream of the 5' major splice donorand continuing to about 14 bases upstream of the gag initiation codon.The number of bases that need to be left out can vary greatly, forexample, the 19 base pair deletion AAAAATTTTGACTAGCGGA deletion in HIV-1(nucleotides 301-319) is sufficient to result in loss of packagingability (See FIG. 1). However, even smaller deletions in this regionshould also result in loss of packaging efficiencies. Indeed, it isexpected that a deletion as small as about 5 base pairs in this regionshould remove packaging ability. Thus the size of a particular deletioncan readily be determined based upon the present disclosure by theperson of ordinary skill in the art.

The vector should contain an HIV nucleotide segment containing asufficient number of nucleotides corresponding to nucleotides of the HIVgenome to express functional HIV gene products, but as aforesaid, shouldnot contain a sufficient number of nucleotides corresponding to theregion between the 5' major splice donor and the gag gene initiationcodon to permit efficient packaging of the viral RNA into virions. Inusing these vectors to establish HIV packaging defective cell lines itis preferred that such cell lines do not produce any infectious HIV.Although a cell line transformed by these packaging deficient vectorswould have low infectivity because the cells are packaging defective,some RNA can still be packaged into the virion. Accordingly, it ispreferable that the HIV nucleotide segment does not correspond to theentire HIV genome so that if some of the viral RNA is packaged into thevirion, what is packaged will not be a replication competent virus.

Preferably, one would want to have at least two different vectors, eachcontaining a different portion of the HIV genome and also not containingthe sequence necessary for viral packaging. Then by co-transfecting acell with each vector the cell would still be able to express all theHIV structural proteins and produce virions. In one preferred embodimentthe vector would not contain sequences corresponding to an HIV LTR butwould contain sequences corresponding to a promoter region and/oranother genome's polyadenylation sequences. Selection of particularpromoters and polyadenylation sequences can readily be determined basedupon the particular host cell. Preferably, the LTR which the sequencesdo not correspond to is the 3' LTR. For example, see FIG. 3.

In one preferred embodiment one vector would include sequencespermitting expression of HIV proteins upstream of env and the secondvector would permit expression of the remaining proteins. For example,one vector would contain an HIV nucleotide segment corresponding to asufficient number of nucleotides upstream of the gag initiation codon tothe env gene sequence to express the 5'-most gene products. The othervector would contain an HIV nucleotide segment corresponding to asufficient number of nucleotides downstream of the gag gene sequence andincluding a functional env gene sequence. Such vectors can be chemicallysynthesized from the reported sequences of the HIV genomes or derivedfrom the many available HIV proviruses, by taking advantage of the knownrestriction endonuclease sites in these viruses by the skilled artisanbased upon the present disclosure (FIG. 3). Preferably, one would alsoadd a different marker gene to each vector, i.e., co-transfect apreselected cell line with these different vectors and by looking for acell containing both markers, one would have a cell line that has beenco-transfected with the two vectors. Such a cell would be able toproduce all of the HIV proteins. However, although virions would beproduced, the RNA corresponding to the entire viral sequences would notbe packaged in these virions. One can use more than two vectors ifdesired, e.g. a gag-pol vector, an env vector and a vif/vpu vector.

For example, one could have a vector comprising nucleotidescorresponding to a sufficient number of nucleotides of an HIV LTR at the5' end to result in a functional LTR, (preferably it corresponds to the5' LTR), nucleotides corresponding to the rev gene and the env genedownstream of the LTR, and at the 3' end, where the sequences do notcorrespond to another LTR, the sequences correspond to polyadenylationsequences, such as polyadenylation sequences corresponding to the SV40virus (e.g., pSVIIIenv 3-2 in FIG. 3). A second vector would containother HIV or SIV genes and contain a deletion in the packaging sequenceand a deletion for the env gene. This vector would also not have a 3'LTR, but would have a polyadenylation sequence. For example, one couldhave a vector which would not contain a sufficient number of nucleotidescorresponding to HIV packaging sequence to package HIV RNA, but wouldcontain a nucleotide segment corresponding to a sufficient number ofnucleotides corresponding to a sufficient number of nucleotides of theHIV gag and pol genes to express functional gag and pol products.Preferably, this vector would also contain a sufficient number ofnucleotides corresponding to a functional tat gene. The vector would notcontain a sufficient number of nucleotides to encode a functional envprotein. More preferably, the vector would also contain nucleotidescorresponding to other HIV regulatory genes to express functional geneproducts, such as vpr, vpu, vif, etc. Other combinations of vectors canalso be prepared. For example, a vector that does not contain asufficient number of nucleotides to correspond to a functional gag gene,but would have a sufficient number of nucleotides to correspond tofunctional pol and env genes, other vectors include one that does notcontain a sufficient number of nucleotides to encode a functional polprotein, but would have a sufficient number of nucleotides to encode adifferent functional HIV protein, etc.

As used herein, the term a sufficient number of nucleotides permitsadditions, deletions and substitutions as long as the claimed functionalability is not lost. For example, if one is referring to the functionalability of packaging HIV RNA then the resultant vector must have asequence that can package HIV RNA. =HIV-1 can be pseudotyped with theenvelope glycoproteins of other viruses. [(Lusso, P., et al., Science247:848-851 (1990)]. Consequently, one can prepare a vector containing asufficient number of nucleotides to correspond to a functional env genefrom a different retrovirus. Preferably, the 5' LTR of this vector wouldbe of the same genome as the env gene. Such a vector could be usedinstead of an env packaging deficient vector to create virions. By sucha change, the resultant vector system can be used in a wider host range.

Virtually any cell line can be used. Preferably, one would use amammalian cell line, for example, CV-1, Hela, Raji, RD, SW480 or CHOcell lines.

In order to increase production of the viral cellular products, onecould use a different promoter than the 5' LTR, i.e., replace the 5' LTRwith a promoter that will preferentially express genes under its controlin a particular cell line. For example, the CMV promoter willpreferentially express genes in CV-1 or Hela cells. The particularpromoter used can be readily determined by the person of ordinary skillin the art, based upon the particular host cell line to be used.

In order to increase the level of viral cellular products one can alsoadd enhancer sequences to the vector to get enhancement of the HIV LTRand/or promoter. Particular enhancer sequences can readily be determinedby the person of ordinary skill in the art depending upon the host cellline.

One can also add vectors that express viral enhancer proteins, such asthose of herpes virus, hepatitis B virus, which act on HIV LTRs toenhance the level of virus product, or cellular transactivator proteins.Cellular transactivation proteins include NF κ-B, UV light responsivefactors and other T cell activation factors well known to the person ofordinary skill in the art.

By using a series of vectors that together would contain the completeHIV genome, one can create cell lines that produce a virion that isidentical to the HIV virion, except that the virion does not contain theHIV RNA. The virions can readily be obtained from these cells. Forexample, the cells would be cultured and supernatant collected.Depending upon the desired use the supernatant containing the virionscan be used or these virions can be separated from the supernatant bystandard techniques. Typically, this would include gradiantcentrifugation, filtering, etc.

These attenuated virions would be extremely useful in preparing avaccine. The virions can be used to generate an antigenic response tothe HIV virions and because these virions are identical to the actualHIV virions, except that the interior of these virions do not containthe viral RNA, the vaccine created should be particularly useful.

These virions can also be used to raise antibodies to the virion thatcan then be used for a variety of purposes, e.g. screening for thevirion, developing target system for the virions, etc.

Additionally, these HIV packaging deficient cell lines can be extremelyuseful as a means of introducing a desired gene, for example, aheterologous gene into mammalian cells.

These virions could be used as an extremely efficient way to packagedesired genetic sequences into target cells infectable by HIV. Thiswould be done by preparing a vector containing a nucleotide segmentcontaining a sufficient number of nucleotides corresponding to thepackaging nucleotides of the HIV virus (HIV packaging region), apredetermined gene, and flanking the packaging sequence and thepredetermined gene with sequences corresponding to a sufficient numberof sequences from within and near the LTRs for packaging, reversetranscription, integration and gene expression. The packaging regionused would preferably correspond to at least the region between the 5'major splice donor and just upstream of the gag initiation codon, morepreferably the region between the 5' major splice donor and the Bal Isite (2202 in HIV-1) in the gag gene.

For example, a sufficient number of HIV-1 sequences to be packaged,reverse transcribed, integrated and expressed in the target cells wouldinlclude the U3, R and U5 sequences of the LTRs, the packagingsequences, and some sequences flanking the LTRs (required for reversetranscription). From the 5' LTR, the R and U5 regions would be includedwhich, in HIV-1, extend from +1 to 183. The sequences flanking the 5'LTR necessary for reverse transcription and packaging would extend from183 to about 335. Although not wishing to be bound by theory, applicantsbelieve the inclusion of additional sequences from the gag gene in thevector (up to the Bal I site, nucleotide 2202) should enhance packagingefficiency. The regions from the 3' LTR and the immediate flankingsequences to be included extend from about 8645 to about 9213 (U3 and Rregions). Analagous regions would be included in a vector based uponHIV-2 or SIV.

When this vector is used to transfect one of the HIV packaging deficientcells, it is the nucleotide sequence from this vector that will bepackaged in the virions. These "HIV packaged" genes could then betargeted to cells infectable by HIV. This method of transformation isexpected to be much more efficient than current methods. Further, byappropriate choice of genes, one could also monitor the method of HIVinfection.

For example, the vector could contain a sufficient number of nucleotidescorresponding to both 5' and 3' LTRs of HIV-1, HIV-2 or SIV to beexpressed, reverse transcribed and integrated, a sufficient number ofnucleotides corresponding to an HIV packaging sequence to be packaged,for example a segment between the 5' major splice donor and justupstream of the gag initiation codon (e.g., nucleotide 381). The vectorwould also contain a sufficient number of nucleotides of the gene whichis desired to be transferred to produce a functional gene (e.g., genesegment). The gene can be any gene desired, for example, the gene forneomycin phosphotransferase (Neo^(R)). More preferably, the "gene" wouldexpress a product that adversely affects HIV replication or integrationsuch as a trans-dominant inhibitor, anti-sense RNAs, catalytic RNAs orsoluble CD4 derivative. With such a gene, the vector of the presentinvention can be used to target HIV target cells. One would preferablyinclude a promoter for the desired gene, although the LTR sequence,itself, can serve as a promoter. Virtually any promoter can be used.Preferably, one would use a promoter that would facilitate expression ofthe gene in the host cell to which the gene is to be transfered.Preferred promoters include viral promoters, such as SL-3, murineretroviral LTR, etc. Enhancers sequences are also preferably used in thevector. One would also preferably include polyadenylation sequences forthe gene. One can use any polyadenylation sequence, for example, thesequences corresponding to SV-40 polyadenylation sequences. The desiredgene can be inserted in the present vector, in either the sense oranti-sense orientation with respect to the LTRs. The vector can containmore than one gene or pseudogene sequence, permitting the expression ofmultiple genes of interest.

This vector can preferably be used with the packaging-defective vectorsdescribed above. In such a situation, one preferably uses HIV LTRs inthe vector corresponding to the genome of the package-deficient vectorto facilitate packaging efficiency. However, in addition to use with apackaging-deficient virus, this vector can also be used with helpervirus for gene transfer. For example, when one wants to deliver a geneto treat an individual infected with AIDS, one could insert this vectorinto that individual and it would be incorporated into HIV virions beingproduced in that individual. This would facilitate the delivery of thedesired gene to the appropriate target cell. Accordingly, one could usethis to deliver trans-dominant inhibitors, anti-sense RNAs, catalyticRNAs, or soluble CD4 derivatives which are also aimed at inhibitingHIV-1 functions critical for viral replication. One could also deliverthis material by using the packaging-defective vectors described hereinor such packaging defective vectors in combination with the HIV virus inan infected individual.

Additionally, these HIV packaging defective cell lines can be used tostudy various stages of the HIV life cycle, both in vivo and in vitrosystems by a system because the cells will express HIV cellularproteins, but will not package the RNA.

The present invention is further illustrated by the following examples.These examples are provided to aid in the understanding of the inventionand are not to be construed as limitation thereof.

The region between the HIV-1 5' LTR and the gag gene is shown in FIG. 1which shows the 5' major splice donor (SD) and site of deletion in avector described below, pHXBΔP1. A 19 base-pair deletion in this regionwas created in an infectious HIV-1 proviral clone contained on theplasmid pHXBc2 of Fisher, A. G., et al, Nature 316: 262-265 (1985). Thisplasmid also contains an SV40 origin of replication to allow efficientgene expression in COS-1 cells. The mutation was produced by thesite-directed mutagenesis as described in Kunkel, T. A., et al, Methodsin Enzymology 154, 367-382 (1987), and the sequence confirmed by DNAsequencing (Sanger, F., et al, Proc. Natl. Acad. Sci. 74:5463-5467(1977). The mutated plasmid was designated pHXBvP1.

An additional out-of-frame deletion between the Bgl II sites(nucleotides 6620 to 7199) was created in the env gene. See FIG. 2b.Then, polyadenylation signals from SV40 were substituted for the 3' endof the pHXBΔP1 provirus beginning at the BamHI site (nucleotide 8053) toprepare HXBΔP1Δenv. The pSVIIIenv3-2 plasmid encodes the HIV-1 env andenv genes, with polyadenylation signals derived from SV40. In thepSVIIIenv 3-2 plasmid, the rev and env genes of HIV-1 are under thecontrol of the HIV-1 LTR. The construction of this vector has beenpreviously described. (See Sodroski, et al., Nature 321:412-417 (1986)and Sodroski, et al., Nature 322:470-474 (1986), which are incorporatedherein by reference. The maps of the relevant portions of the plasmidsused are shown in FIG. 2. For the HXBΔP1 and HXBΔP1Δenv vectors, thepositions of the HIV-1 genes are shown along with the location of anineteen base pair deletion previously described (Δ19 bp). And forHXBΔP1Δenv and pSVIIIenv 3-2, the rev responsive element (RRE), and theSV40 polyadenylation signal (SV40 polyA) are shown.

COS-1 cells were maintained in Dulbecco's modified Eagle's medium DMEM(Hazelton Biologics, Lenexa) supplemented with 10% fetal calf serum(Gibco, Long Island, N.Y.) and antibiotics. Jurkat cells were maintainedin culture in RPMI 1640 with 10% fetal calf serum and antibiotics.Jurkat cells were preventatively treated for mycoplasma with B. M.Cyclin I and II and human serum two weeks prior to infection. The HXBc2(gag⁺, pro⁺, pol⁺, vif⁺, vpr⁻, vpu⁻, tat⁺, rev⁺, env⁺, nef⁻) proviruswas used in all plasmids was used in all plasmid and vector constructs.

To evaluate the effect of the mutation on viral protein expression andvirion production, COS-1 cells were transfected with the pHXBc2 andpHXBΔP1 plasmids by the DEAE-dextran procedure [Lopata et al, Nucl.Acids Res. 12:5707-5717 (1984); Queen and Baltimore, Cell 33:741-748(1983); (Sodroski, J., et al, Science 231:1549-1553 (1986) which areincorporated herein by reference]. COS-1 cell lysates and supernatantsradiolabelled with ³⁵ S-cysteine (Sodroski, J., et al, Science 231,supra) at 48 hours after transfection were precipitated with 19501 AIDSpatient serum. The overall level of viral protein detected in celllysates was comparable for the vectors containing the wild-type HXBc2and the HIV packaging defective HXBΔP1. See FIG. 5A, which showsimmunoprecipitation of ³⁵ S-labelled viral proteins from COS-1 celllysates (lanes 1-3) or supernatants (lanes 4-6) with 19501 patientserum, after transfection with no DNA (lanes 1 and 4), 10 μg pHXBc2(lanes 2 and 5) or 10 μg pHXBΔP1 (lanes 3 and 6). The overall level ofviral protein detected in cell lysates was comparable for cellstransfected by either HXBc2 or HXBΔP1. The level of viral proteinsprecipitated from the supernatants of COS-1 cells was slightly less withHXBΔP1 than with HXBc2. The amount of reverse transcriptase (RT)activity measured in the supernatants of COS-1 cell transfected bypHXBΔP1 was 60% of that measured in cells transfected with the HXBc2vector (data not shown). COS-1 cells transfected with pHXBΔP1 were fixed48 hours following transfection and examined by electron microscopy.Viral particles, including budding forms, of normal HIV-1 morphologywere observed. FIG. 5B is an electron micrographs of COS-1 cellstransfected with PHXBΔP1 showing virus particles of normal HIV-1morphology.

To evaluate the effect of the HXBΔP1 mutation on HIV-1 replication,supernatants from COS-1 cells transfected with pHXBc2 and pHXBΔP1 werefiltered (0.2μ) and RT measured. Supernatants containing equal amountsof RT activity of mutant and wild-type viruses were added to Jurkathuman T lymphocytes. The Jurkat cultures along with a mock-infectedculture were maintained with medium changes every three days. Atintervals aliquots of Jurkat cells were labelled and assessed forexpression of HIV-1 proteins by immunoprecipitation with 19501 AIDSpatient serum. FIG. 6 shows immunoprecipitation of labelled viralproteins from Jurkat T cell lysates (lanes 1-3, 7-9 and 13-15) orsupernatants (lanes 4-6, 10-12 and 16-18) exposed to supernatants fromCOS-1 cells that were mock transfected (lanes 1, 4, 7, 10, 13 and 16),pHXBc2 (lanes 2, 5, 8, 11, 14 and 17), or transfected with pHXBΔP1(lanes 3, 6, 9, 12, 15 and 18). The Jurkat cells were examined at day 7(lanes 1-6), day 14 (lanes 7-12) and day 21 (13-18) following infection.Jurkat cultures exposed to HXBΔP1 exhibited marked delays in and lowerlevels of viral protein production relative to those exposed to pHXBc2,the wild-type virus. Virus replication in human T lymphocytestransfected by HXBΔP1 is thus seen to be significantly attenuatedcompared with cells transfected by HXBc2.

Supernatants from the above cultures were 0.2μ-filtered and equivalentamounts of reverse transcriptase activity pelleted by centrifugation at12000 xg for one hour at 20° C. Viral pellets were lysed by NP40 in thepresence of vanadyl ribonucleotides and dilutions of virus dot-blottedonto nitrocellulose filters. Some samples were treated with sodiumhydroxide (5M at 60° C. for 15 minutes) prior to dot-blotting. Filterswere hybridized with a DNA probe consisting of HIV-1 gag and env genesequences, washed and autoradiographed as previously described inManiatis, T., et al, Molecular Cloning, Cold Spring Harbor Laboratory,(1982). For the wild-type HXBc2 virus, a signal specific for RNA couldbe detected after blotting 1000 reverse transcriptase units of filteredsupernatant (not shown). For the HXBΔP1, even 5×10⁴ reversetranscriptase units of supernatant gave no detectable signal. FIG. 7 isan RNA dot blot without (column 1) and with (column 2) sodium hydroxidetreatment following blotting of filtered supernatants from the Jurkatcultures. The supernatants contained a reverse transcriptase activity of5×10⁴ cpm of HXBΔP1 (row A), 5×10⁴ cpm of HXBc2 (row B), or 1×10⁵ cpm ofHXBc2 (row C). These results indicate that the efficiency of packagingvirus-specific RNA into virions for cells transfected with a packagingdefective viral vector according to the present invention is less than2% of the wild-type virus.

The results indicate that the region between the 5' LTR and gag gene ofHIV-1 is important for packaging viral RNA into virions. A mutation inthis region exhibits minimal effects on the ability of the provirus toproduce proteins and virion particles following transfection, butmarkedly decreases the level of virion RNA and attenuates virusreplication in a human CD4-positive lymphocyte line. HIV-1 replicates incultured CD4-positive cells via cell-free transmission and cell-to-celltransmission, the latter involving the contact of infected anduninfected cells [Fisher, A. G., et al, Nature 316:262-265 (1985),Sodroski, J., et al, Science 231:1549-1553 (1986), Strebel, K., et al,Nature 328:728-730 (1987)].

Vectors based on HIV-1 packaging sequences were constructed.

The pHVB(SL3-Neo)sense ##STR1## and pHVB (SL3-Neo)anti-sense ##STR2##plasmids contain the coding sequence of the neo gene under the controlof the SL3-3 murine leukemia virus LTR, with polyadenylation signalsderived from SV40. The pHVB(SL3-Neo)sense and pHVB(SL3-Neo) anti-senseplasmids contain complete 5' and 3' HIV-1 LTRs and flanking viralsequences nucleotides 183-381 near the 5' LTR and nucleotides 8504-8661adjacent to the 3' LTR. A unique Bam HI site was inserted at theboundaries of the major deletion in the HXBc2 provirus (nucleotides 382to 8593), and the SL3 LTR-Neo transcription unit was cloned into thissite in either the sense ##STR3## or antisense ##STR4## orientation withrespect to the HIV-1 LTRs. All of the plasmids contained SV40 origins ofreplication. See FIG. 2. These vectors include the 19 base pair sequenceshown by deletion to be important for packaging viral RNA. The vectorscannot encode any of the HIV-1 gene products. The vectors contain aninsert in which the SL3-3 murine retroviral LTR, which functions as anefficient promoter in T lymphocytes, promotes the expression of theneomycin phosphotransferase (Neo^(R)) gene. The polyadenylation signalsfor the Neo transcript are provided by sequences from SV40. Numbersabove the plasmid in FIG. 2 indicate the nucleotide of the HXBc2sequence that form the boundaries of provirus/insert. The SL3-3 murineleukemia virus LTR is indicated SL3. The position of the major 5' splicedonor (SD) and gag gene initiation codon (gagATG) are shown in thefigure.

50% confluent COS-1 cells were transfected with plasmid DNA for thesystems HXBΔP1+HVB (SL3-Neo)sense; HXBΔP1+HVB(SL3-Neo)anti-sense,HVB(SL3-Neo)sense +pHXBΔP1Δenv +pSVIIIenv 3-2; andHVB(SL3-Neo)anti-sense+pHXBΔP1Δenv +pSVIIIenv 3-2 using 10 μg/ml of eachplasmid by the calcium phosphate method [Chen, C., et al., Mol. Cell.Biol. 7:2745-2752 (1987)] to generate recombinant viruses. DMEMcontaining FCS and antibiotics was placed in COS-1 cell cultures 12hours after transfection. COS-1 supernatants were harvested and filteredthrough a 0.2 μm filter (Millipore) 72 hours post transfection. The p24level in the COS-1 supernatants was determined by a p24 radioimmunoassay(Dupont).

Jurkat cells to be infected with virus were seeded into 6-well cultureplates at a 2.5×10⁵ cells per well in 2.5 ml of complete medium.Ten-fold serial dilutions of transfected COS-1 supernatants were thenapplied to each well and the virus allowed to absorb to the Jurkat cellsat 37° C. for 4 hours. After this the Jurkat cells were pelleted andresuspended in complete medium. Twenty-four hours later the medium wasreplaced by complete medium containing G418 (Gibco, N.Y.) at an activeconcentration of 0.8 mg/ml and the cells dispensed into 24-well cultureplates at 1.0×10⁴ cells per well. Culture medium was changed every 4days. Positive wells containing clusters of viable G418-resistant cellswere identified and counted in cultures 18 days post-infection. Levelsof p24 in infected Jurkat cell culture medium were determined at 5, 10,and 20 days post-infection by radioimmunoassay. The level of syncytiumformation by infected Jurkat cells was also determined in cultures 5 to20 days post-infection.

To clone the G418-resistant Jurkat cells, the cells were washed inphosphate-buffered saline and diluted to a concentration of 0.5 viablecells per 100 μl medium. Then 100 μl of the cell suspension wasdispensed into each well of a 96-well culture plate. Wells containingsingle cells were identified by phase contract microscopy and individualcells were expanded to 10⁷ cells in complete medium containing 0.8 mg/mlG418.

Genomic DNA was prepared from clones, digested with SacI and Southernblotted as previously described by Southern, E. M, J. Mol. Biol.98:503-517 (1975). Southern blots were hybridized to a 3.3 Kb fragmentcontaining the SL3 LTR, neo gene and SV40 polyadenylation sequences thathad been labelled by random priming with oligonucleotides. Southernblots were washed under conditions of high stringency.

The pHXBΔP1 plasmid and the HIV-1 vectors were cotransfected into COS-1cells as described above. Table 1 shows that gag p24 protein of HIV-1was detectable in the supernatant of these transfected cells on thethird day after transfection. The filtered COS-1 supernatants wereserially diluted and incubated with Jurkat lymphocytes, which wereselected for G418-resistance. The number of G418-resistant Jurkat cellsgenerated (Table 1) ranged from 10² to 10⁵ per milliliter of COS-1supernatant, with the HVB(SL3-Neo)anti-sense vector yielding highertiters than the HVB(SL3-Neo)sense vector. No G418-resistant Jurkat cellswere generated following incubation with supernatant derived from COS-1cells transfected with no DNA, the vectors alone, or the pHXBΔP1 plasmidalone.

The HXBΔP1 provirus is not completely replication defective. Thus, theproduction of viral p24 antigen and the formation of syncytia wereexamined to determine the amount of infectious virus in theG418-resistant Jurkat cells. FIG. 4 shows that HIV-1 p24 antigen wasdetectable in the supernatants of the Jurkat cultures. Syncytiumformation was visible and increased with time in these cultures,indicating the expression of the HIV-1 envelope glycoproteins in thetarget cells. The induction of significant cytophathic effect in thesecultures made cloning of the G418-resistant Jurkat cells difficult, andfurther suggested the presence of replication-competent viruses in thetarget cells. See also Table 2.

                  TABLE 1                                                         ______________________________________                                        Viral p24 Antigen Expression And Recombinant                                   Viral Titers In Supernatants of Transfected COS-1 Cells.                                                   G418-resistant titers                                                          Transfected DNA p24(ng/ml).sup.a cfu/ml).su                                  p.a                                             ______________________________________                                        None            0         0                                                     pHVB(SL3-Neo)sense 0 0                                                        pHVB(SL3-Neo)anti-sense 0 0                                                   pHXBΔP1 0.6 0                                                           pHXBΔP1 + 6.1 5 × 10.sup.2                                        pHVB(SL3-Neo)sense                                                            pHXBΔP1 + 22.0 1 × 10.sup.4                                       pHVB(SL3-Neo)anti-sense                                                       None 0 0                                                                      pSVIIIenv3-2 0 0                                                              pHXBΔP1Δenv   5.6 0                                               pHXBΔP1Δenv +  2.5 1 × 10.sup.5                             pSVIIIenv3-2 +                                                                pHVB(SL3-Neo))sense                                                           pHXBΔP1Δenv +  1.1 1 × 10.sup.4                             pSVIIIenv3-2 +                                                                pHVB(SL3-Neo)anti-sense                                                     ______________________________________                                         .sup.a Values shown are the mean results of three experiments.           

Two other plasmids described above encoding the HIV-1 proteins wereutilized instead of the HXBΔP1 provirus. The pHXBΔP1Δenv plasmid isidentical to the pHXBΔP1 plasmid except that the provirus in the formercontains a deletion in the env gene and contains a polyadenylationsignal from SV40 in place of the 3' LTR. The pSVIIIenv3-2 plasmidencodes both rev and env genes of HIV-1 under the control of the HIV-1LTR, with polyadenylation signals derived from SV40. When these twoplasmids were cotransfected into COS-1 cells along with thepHVB(SL3-Neo) sense or pHVB(SL3-Neo) anti-sense plasmids by the methodsdescribed above, p24 gag antigen could be detected in the supernatantsthree days after transfection (Table 1). Incubation of thesesupernatants with Jurkat lymphocytes and selection of the Jurkat cellswith G418 indicated that the level of recombinant virus ranged form 10⁴to 10⁶ colony-forming units per millilliter of filtered supernatant. Inthese experiments, no significant differences were noted betweenexperiments, and no significant difference was noted between theHVB(SL3-Neo)sense and HVB(SL3-Neo)anti-sense vectors in three separateexperiments.

These G418-resistant Jurkat cells were cloned and used for isolation ofDNA. The genomic DNA was digested with SacI, which cuts the vectors oncein each of the HIV-1 LTRs to produce a 3.3 Kb fragment. A fragment whichcontains sequences derived from the SL3-3 LTR, the coding sequences ofthe Neo^(R) gene, and SV40 polyadenylation signals was used as a probe.Clones derived from both the HVB(SL3-Neo)sense andHVB(SL3-Neo)anti-sense vectors demonstrated a single hybridizing band at3.3 Kb that was not seen in control Jurkat cells. See FIG. 8. Thisdemonstrates that the HVB(SL3-Neo)sense and HVB(SL3-Neo)anti-sensesequences had been transferred without rearrangement or recombinationinto the G418-resistant Jurkat cells.

p24 gag protein was measured in cell supernatants and syncytia werescored up to 40 days following the initial infection. In all of theclones examined, no syncytia were observed and p24 antigen wasundetectable. See Table 2. This indicates that no replication-competentvirus was present in these G418-resistant Jurkat cells.

                  TABLE 2                                                         ______________________________________                                        Syncytium Formation in G418-resistant Jurkat Cultures.sup.a                                      Days After Infection                                       DNA Transfected into COS-1 Cells                                                                 5     10      20   40                                      ______________________________________                                        pHXBΔP1 +    -     +       +++  ND                                        pHVB(SL3-Neo)sense                                                            pHXBΔP1 + - - ++ ND                                                     PHVB(SL3-Neo)anti-sense                                                       pHXBΔP1Δenv +  - - - -                                            pSVIIIenv3-2 +                                                                pHVB(SL3-Neo)sense                                                            pHXBΔP1Δenv +  - - - -                                            pSVIIIenv3-2 +                                                                pHVB(SL3-Neo)anti-sense                                                     ______________________________________                                         .sup.a Syncytia were scored according to the following criteria: -, no        syncytia observed; +, 1-5 syncytia/hpf; ++, 5-10 syncytia/hpf; +++,           greater than 10 syncytia/hpf (hpf = high power field).                   

The results suggest that the packaging-defective HXBΔP1 provirus canprovide trans-acting viral functions required for the transfer of aHIV-1 vector to Jurkat lymphocytes. The HIV-1 LTRs and the immediateflanking sequences, including the sequences defined to be important forpackaging viral RNA, appear to be sufficient to allow packaging, reversetranscription and integration. By providing the trans-acting functionson two separate plasmids, each lacking a 3' LTR, the transfer of vectorsequences occurs in the apparent absence of replication-competent virus.The titers of recombinant virus in this helper virus-free context appearto be improved relative to those observed in the presence ofreplication-competent virus, probably because of the induction ofsignificant cytopathic effects by the latter. The higher titer observedfor the HVB(SL3-Neo)anti-sense vector relative to the HVB(SL3-Neo)sensevector in the presence of replication-competent virus may in part relateto a suppresive effect of anti-sense read-through transcripts from theSL3 promoter on helper virus replication.

The HIV-1 vector described herein provides a simple, efficient means ofintroducing individual genes of interest into potential HIV-1 targetcells. In light of recent observations demonstrating that HIV-1 can bepseudotyped with the envelope glycoproteins of other viruses, increasingthe host range of these vectors is feasible. Given the ability of thewild-type HIV-1 genome to encode multiple gene products, these vectorsare readily adaptable for the expression of multiple genes of interestin the target cells.

It is evident that those skilled in the art, given the benefit of theforegoing disclosure, may make numerous modifications thereof anddepartures from the specific embodiments described herein, withoutdeparting from the inventive concepts and the present invention is to belimited solely by the scope and spirit of the appended claims.

We claim:
 1. At least two vectors, wherein there is at least a firstvector and a second vector, said vectors comprising:(a) a first HIVvector, containing nucleotides sufficient to encode an HIV gag protein,wherein said nucleotides are operably linked to a promoter and apolyadenylation sequence; (b) a second vector, nucleotides sufficient toencode a functional env gene of a virus other than HIV, wherein saidnucleotides are operably linked to a promoter and a polyadenylationsequence;wherein the vectors, when ex-pressed in combination, form apseudotyped virion containing an envelope protein other than the HIV envprotein around an HIE capsid; and wherein said vectors do not contain asufficient number of nucleotides to encode the HIV gag, env and polproteins on a single vector; and wherein said vectors do not containnucleotides of the HIV genome, referred to as the HIV packaging segment,between the 5' major splice donor and the gag gene sufficient toeffectively package HIV RNA; and wherein said vectors, when expressed incombination, do not form a replication competent virus.
 2. The at leasttwo vectors of claim 1, wherein said vectors, when expressed incombination, express an HIV pol protein.
 3. The at least two vectors ofclaim 2, wherein nucleotides sufficient to encode said HIV pol proteinare on said first vector.
 4. At least three vectors, wherein there is atleast a first, second and third vector, said vectors comprising:(a) afirst vector, containing nucleotides sufficient to encode an HIV gagprotein, operably linked to a promoter and a polyadenylation sequence;(b) a second vector, containing nucleotides sufficient to encode afunctional env gene of a virus other than HIV, operably linked to apromoter and a polyadenylation sequence; and (c) a third vectorcontaining a gene or interest, operably linked to a promoter and an HIVpacking sequence necessary to package HIV RNA into HIV virions;whereinthe first and second vector, when expressed in combination, form apseudotyped virion containing an envelope protein other than the HIV envprotein around an HIV capsid; and wherein said vectors do not contain asufficient number of nucleotides to encode the HIV gag, env and polproteins on a single vector; and wherein said first and second vectorsdo not contain nucleotides of the HIV genome, referred to as the HIVpackaging segment, between the 5' major splice donor and the gag genesufficient to effectively package HIV RNA; and wherein said vectors,when expressed in combination, do not form a replication competentvirus.
 5. The at least three vectors of claim 4, wherein said first andsecond vector, when expressed in combination express an HIV pol protein.6. The at least three vectors of claim 5, wherein nucleotides sufficientto encode said HIV pol protein are on said first vector.
 7. At least twovectors, wherein there is at least a first vector and a second vector,said vectors comprising:(a) a first HIV vector, containing nucleotidessufficient to encode an HIV gag protein, wherein said nucleotides areoperably linked to a promoter and a polyadenylation sequence; (b) asecond vector, nucleotides sufficient to encode a functional env gene ofa virus other than HIV, wherein said nucleotides are operably linked toa promoter and a polyadenylation sequence;wherein the vectors, whenexpressed in combination, form a pseudotyped virion containing anenvelope protein other than the HIV env protein around an HIV capsid;(c) wherein said first or said second vector contains nucleotidessufficient to encode an HIV pol protein, wherein said nucleotides areoperably linked to a promoter and a polyadenylation sequence; andwhereinsaid vectors do not contain a sufficient number of nucleotides to encodethe HIV gag, env and pol proteins on a single vector; and wherein saidvectors do not contain nucleotides of the HIV genome, referred to as theHIV packaging segment, between the 5' major splice donor and the gaggene sufficient to effectively package HIV RNA; and wherein saidvectors, when expressed in combination, do not form a replicationcompetent virus.
 8. At least three vectors, wherein there is at least afirst vector, a second vector, and a third vector, said vectorscomprising:(a) a first HIV vector, containing nucleotides sufficient toencode an HIV gag protein, operably linked to a promoter and apolyadenylation sequence; (b) a second vector containing nucleotidessufficient to encode a functional env gene of a virus other than HIV,operably linked to a promoter and a polyadenylation sequence; (c) athird vector, containing nucleotides sufficient to encode an HIV polprotein, operably linked to a promoter and to a polyadenylationsequence;wherein the vectors, when expressed in combination, form apseudotyped virion containing an envelope protein other than the HIV envprotein around an HIV capsid; wherein said vectors do not contain asufficient number of nucleotides to encode the HIV gag, env and polproteins on a single vector; and wherein said vectors do not containnucleotides of the HIV genome, referred to as the HIV packaging segment,between the 5' major splice donor and the gag gene sufficient toeffectively package HIV RNA; and wherein said vectors, when expressed incombination, do not form a replication competent virus.
 9. At leastthree vectors, wherein there is at least a first, second and thirdvector, said vectors comprising:(a) a first vector, containingnucleotides sufficient to encode an HIV gag protein, operably linked toa promoter and a polyadenylation sequence; (b) a second vector,containing nucleotides sufficient to encode a functional env gene of avirus other than HIV, operably linked to a promoter and apolyadenylation sequence; and (c) a third vector contains a gene orinterest, operably linked to a promoter and an HIV packing sequencenecessary to package HIV RNA into HIV virions; (d) and wherein saidfirst or second vectors contain nucleotides sufficient to encode an HIVpol protein, wherein said nucleotides are operably linked to a promoterand a polyadenylation sequence;wherein the first and second vector, whenexpressed in combination, form a pseudotyped virion containing anenvelope protein other than the HIV env protein around an HIV capsid;and wherein said vectors do not contain a sufficient number ofnucleotides to encode the HIV gag, env and pol proteins on a singlevector; and wherein said first and second vectors do not containnucleotides of the HIV genome, referred to as the HIV packaging segment,between the 5' major splice donor and the gag gene sufficient toeffectively package HIV RNA; and wherein said vectors, when expressed incombination, do not form a replication competent virus.
 10. At leastfour vectors, wherein there is at least a first, second, third, andfourth vector, said vectors comprising:(a) a first vector containingnucleotides sufficient to encode an HIV gag protein, wherein saidnucleotides are operably linked to a promoter and a polyadenylationsequence; (b) a second vector containing nucleotides sufficient toencode a functional env gene of a virus other than HIV, wherein saidnucleotides are operably linked to a promoter and a polyadenylationsequence; and (c) a third vector containing nucleotides sufficient toencode an HIV pol protein, wherein said nucleotides are operably linkedto a promoter and a polyadenylation sequence; and (d) a fourth vectorcontaining a gene of interest, wherein said gene of interest is operablylinked to a promoter and an HIV packing sequence necessary to packageHIV RNA into HIV virions;wherein the first vector, second vector andthird vector, when expressed in combination, form a pseudotyped virioncontaining an envelope protein other than the HIV env protein around anHIV capsid; and wherein said at least first, second, third and fourthvectors do not contain a sufficient number of nucleotides to encode theHIV gag, env and pol proteins on a single vector; and wherein saidfirst, second and third vectors do not contain nucleotides of the HIVgenome, referred to as the HIV packaging segment, between the 5' majorsplice donor and the gag gene sufficient to effectively package HIV RNA;and wherein said at least first, second, third and fourth vectors, whenexpressed in combination, do not form a replication competent virus. 11.The vectors of claim 4, wherein at least one vector is a retroviralvector.